Project Summary The overall aims of this research are to understand the molecular mechanism by which actomyosin motility systems convert chemical energy into mechanical work, and to obtain a precise correlation between the mechanical, biochemical and structural events at the molecular level. Novel methods will be applied to non-muscle myosin molecular motors to probe the relations between biochemical reactions of the contractile proteins, the elementary mechanical steps of the cross-bridge cycle and the corresponding structural motions. Bifunctional, bi- arsenical and quantum rod fluorescent probes will be stably bound with known orientation to the motor domains, light chain subunits, and tails of the motors. The spatial orientation and translational position of these components will be monitored at high time resolution by novel single-molecule polarized fluorescence, total internal reflection (polTIRF) microscopy to determine the dynamics of specific protein structural changes during translocation along actin and under mechanical load. Increased time resolution recently achieved for measuring the rotational, translational, and thermal wobbling motions and will enable detailed events to be detected during the brief period of molecular stepping between stable dwell periods. An infrared optical trap, with high-speed feedback to clamp the actin in place and to rapidly measure the myosin working stroke after actin attachment, will be used to determine the specific relationships between release of ATPase products, phosphate and ADP, strengthening of the actomyosin bond, transition into force generating states, and tilting, resulting in movement of the cargo. The feedback optical trap will be combined with single-molecule polTIRF microscopy to directly evaluate the influence of mechanical stress, strain, and flexibility on stepping rates and protein orientation changes that relate to chemo-mechanical transduction. The energetics and statistics of actin subunit target selection will be determined from the orientation and force dependence of the domain angles, biochemical states and step sizes. The experiments will be carried out on non-muscle myosins isolated from recombinant expression systems. Results from this project should significantly advance knowledge of cell motility processes and thus bring a greater understanding of both normal and pathological states of neuronal and sensory-neural development and many other types of cell motility.